Neurotox Res (2018) 33:213–221 DOI 10.1007/s12640-017-9814-x

REVIEW

L-Serine: a Naturally-Occurring Amino Acid with Therapeutic Potential

J. S. Metcalf1 & R. A. Dunlop1 & J. T. Powell1 & S. A. Banack1 & P. A. Cox1

Received: 2 February 2017 /Revised: 25 August 2017 /Accepted: 5 September 2017 /Published online: 19 September 2017 # Springer Science+Business Media, LLC 2017

Abstract In human neuroblastoma cell cultures, non-human AD Alzheimer’sdisease primates and human beings, L-serine is neuroprotective, acting ALS Amyotrophic lateral sclerosis through a variety of biochemical and molecular mechanisms. ALS/PDC Amyotrophic lateral sclerosis/parkinsonism Although L-serine is generally classified as a non-essential ami- dementia complex no acid, it is probably more appropriate to term it as a ATF4 Activating transcription factor 4 Bconditional non-essential amino acid^ since, under certain cir- ATF6 Activating transcription factor 6 cumstances, vertebrates cannot synthesize it in sufficient quan- ATG8 Autophagy-related gene 8 tities to meet necessary cellular demands. L-serine is BBB Blood brain barrier biosynthesized in the mammalian central nervous system from L-BMAA β-N-methylamino-L-alanine 3-phosphoglycerate and serves as a precursor for the synthesis CNS Central nervous system of the amino acids glycine and cysteine. Physiologically, it has a CSF Cerebralspinalfluid variety of roles, perhaps most importantly as a phosphorylation DRG Dorsal root ganglion site in proteins. Mutations in the metabolic enzymes that syn- eIF2α Eukaryotic initiation factor 2alpha thesize L-serine have been implicated in various human dis- ER Endoplasmic reticulum eases. Dosing of animals with L-serine and human clinical trials ERAD Endoplasmic-reticulum-associated investigating the therapeutic effects of L-serine support the protein degradation FDA’s determination that L-serine is generally regarded as safe FTLD-MND Frontotemporal lobar degeneration (GRAS); it also appears to be neuroprotective. We here consider with motor neuron disease the role of L-serine in neurological disorders and its potential as FTLD-U Frontotemporal lobar degeneration a therapeutic agent. with ubiquitinated inclusions FTDP Frontotemporal dementia with parkinsonism Keywords L-serine . Alzheimer’s Disease . Therapy . GluR Glutamate receptors Neuroprotection . ALS . Neurodegeneration GDP Guanosine diphosphate GCN2 General control nondepressible 2 GRAS Generally regarded as safe GTP Guanosine triphosphate Abbreviations HRI Hepatic heme-regulated inhibitor 3-PGDH 3-phosphoglycerate dehydrogenase Met-tRNAi Initiator methionyl tRNA asc-1, asc-2 Alanine-serine-cysteine transporters IRE1 Inositol-requiring enzyme 1 1or2 mRNA Messenger ribonucleic acid tRNA Transfer ribonucleic acid J. S. Metcalf and R. A. Dunlop contributed equally to this work. NFTs Neurofibrillary tangles NRF-2 Nuclear factor E -related factor 2 * P. A. Cox 2 [email protected]; [email protected] PERK Protein kinase RNA-like endoplasmic reticulum kinase 1 Brain Chemistry Labs, The Institute for Ethnomedicine, PKR Protein kinase R Jackson 83001, WY, USA PSAT Phosphoserine aminotransferase 214 Neurotox Res (2018) 33:213–221

PSP Phosphoserine phosphatase those of L-BMAA-dosed macaques which exhibited neuro- Rpt Proteasome subunit regulatory particle 1 logical deficits (Spencer et al. 1987; Duncan 1992). TDP-43 TAR DNA binding protein of 43 kDa Subsequent studies revealed that much of the L-BMAA pool UPIs Ubiquitin-positive inclusions in foodstuffs such as cycad flour is protein-bound as revealed UPR Unfolded protein response by the liberation of L-BMAA from the protein fraction upon XBP1 X-box binding protein-1 hydrolysis and, therefore, the L-BMAA content in Chamorro XBP1s Spliced X-box binding protein-1 washed cycad flour proved to be much higher than previous studies indicated (Cheng and Banack 2009). Thus, an understanding of the importance of the protein- Discovery of the Therapeutic Potential of L-Serine bound L-BMAA pool emerged, and we began a targeted search to determine if L-BMAA was being misincorporated Our interest in the therapeutic potential of L-serine emerged into proteins, and if so, which canonical amino acid it was from over a decade of research into the neurotoxic non- replacing. This conclusion was confirmed by independent labs protein amino acid, β-N-methylamino-L-alanine (L-BMAA), using different models, finding that L-BMAA could be and its link to neurodegenerative diseases, in particular, misincorporated into peptide chains (Dunlop et al. 2013; Guamanian amyotrophic lateral sclerosis/parkinsonism demen- Glover et al. 2014). However, evidence for misincorporation Escherichia coli tia complex (ALS/PDC). ALS/PDC is a neurodegenerative dis- was not found in (van Onselen et al. 2015) ease with a combination of parkinsonism, amyotrophic lateral suggesting perhaps a bacterial mechanism exists for avoid- sclerosis (ALS) and Alzheimer’s-like dementia associated with ance of L-BMAA misincorporation. neurofibrillary tangles (NFTs) composed of the Meticulous screening of the 20 canonical amino acids used hyperphosphorylated microtubule-associated protein, tau to make human proteins revealed that L-BMAA substitutes (Hirano et al. 1961; Hirano and Zimmerman 1962; for L-serine in the peptide chain in mammalian cell cultures Trojanowskietal.2002). L-BMAA is found in the proteins of (Dunlop et al. 2013). Evidence for such misincorporation is post-mortem brain tissues of Chamorro villagers who died of supported by an independent laboratory investigation using a Guamanian ALS/PDC, but generally not in patients who died cell-free system (Glover et al. 2014). of diseases unrelated to neurodegenerative illness (Cox et al. These observations, together with new research suggest 2003; Murch et al. 2004a) or those who have Huntington’s that L-serine is a neuroprotective agent or a prophylactic inde- disease, which is an autosomal dominant neurodegenerative pendent of BMAA exposure (See Dunlop et al. 2017). This condition (Pablo et al. 2009). review seeks to summarize the role of L-serine in mammalian Observations that L-BMAA is present in high concentra- systems, from development to pathology, and explore ways in tions in the protein-bound fraction of flour samples made from which it might be applied as a therapy. cycad seeds (Murch et al. 2004b; Cheng and Banack 2009), cyanobacteria (Cox et al. 2005; Esterhuizen and Downing 2008), and post-mortem Chamorro brain tissues from Guam L-Serine Metabolism, Pathways of Synthesis, (Murch et al. 2004a) led to the hypothesis that L-BMAA might and Deficiency Disorders be protein associated. Initially, it was argued that villagers in Guam would have to consume over 1000 kg of L-BMAA- L-Serine Metabolism and Bioavailability containing cycad flour per year to get a dose equivalent to L-serine is a polar amino acid critical for cellular metabolism and neurological development and function. Concentrations of L- Table 1 Summary of the evidence for the potential of L-serine as a therapeutic in human disease serine found in human cerebral spinal fluid (CSF) and blood plasma generally decrease with age, suggesting a changing need Disease Reference/clinical trial (NCT) for this amino acid throughout a lifetime (van der Crabben et al. Non-alcoholic fatty liver disease (NCT02599038) 2013). Sources of L-serine include dietary intake, de novo syn- thesis, and recycling of L-serine via cellular protein degradation Alcoholic fatty liver disease Sim et al. 2015 (de Koning et al. 2003). The relative contributions of each of these Hereditary sensory neuropathy Garofalo et al. 2011; (NCT01733407) Type 1 pathways have been reviewed by Kalhan and Hanson (2012). Amyotrophic lateral sclerosis Levine et al. 2017; (NCT01835782) Although considered to be a non-essential amino acid, it Alzheimer’s disease (NCT03062449) might be more appropriate to classify L-serine as a ALS/PDC Cox et al. 2016 Bconditional non-essential amino acid,^ since under certain Ichthyosis/polyneuropathy Catsman-Berrevoets et al. 1997 circumstances vertebrates cannot synthesize it in sufficient quantities to meet necessary cellular demands (de Koning NCT clinical trial at clinicaltrials.gov et al. 2003). For this reason, dietary intake is an important Neurotox Res (2018) 33:213–221 215 source of L-serine, even in healthy individuals, and supple- L-serine deficiencies involving SSP enzymes have been mentation may be useful as a neuroprotective prophylactic. well documented, although 3-phosphoglycerate dehydroge- Studies using in vitro models show that L-serine-starved cell nase (3-PGDH) is most often implicated. While deleting 3- cultures significantly upregulate enzymes associated with cellu- PGDH in mice is embryonically fatal (Furuya et al. 2008), lar L-serine production (Lockart and Eagle 1959). Exogenously human patients with 3-PGDH deficiencies exhibit hypo- supplied L-serine is required by rapidly proliferating cells, which myelination, microcephaly, seizures, and profound mental use it for one-carbon metabolism and the folate cycle (Yang and and psychomotor retardation (Jaeken et al. 1996a). Vousden 2016). Depriving cells of access to L-serine has been Supplementation with L-serine and glycine produces modest shown to arrest growth and development of some tumor cells improvements in patient outcomes, reducing seizure activity. both in vivo and in vitro (Maddocks et al. 2012). Some cancer However, L-serine treatment of infants had no effect on psy- cells also exhibit nuclear factor E2-related factor 2 (NRF-2) and chomotor and mental retardation (Jaeken et al. 1996b). activating transcription factor 4 (ATF4)-mediated oxidative Deficiencies in phosphoserine aminotransferase (PSAT) stress during the switch to de novo L-serine synthesis (Hartetal.2007) and phosphoserine phosphatase (PSP) (DeNicola et al. 2015). A cancer-associated transcription factor, (Jaeken et al. 1996b), although much less documented, have cMyc, induces all three enzymes involved in de novo L-serine also been observed. Certainly, with respect to timing of L- synthesis (see Fig. 1); further, suggesting nutrient-deprived tu- serine administration, pre-natal or Bat birth^ supplementation mor cells are dependent upon the endogenous serine biosynthe- with L-serine may help with patient outcomes (Hart et al. sis pathway [(SSP; (Sun et al. 2015)]. 2007). This most likely relates to CNS L-serine concentrations The importance of L-serine supply and synthesis in the being restored to adequately higher levels during a time of central nervous system (CNS) was highlighted when it was intense CNS development and cellular proliferation. discovered that the enantiomer, D-serine, plays an important Defects in L-serine biosynthesis or transport produce pro- role in neurotransmission and activation of glutamate recep- found developmental disorders. Neu-Laxova syndrome is a tors (GluR) (Wolosker et al. 1999). L-serine crosses the blood- rare autosomal-recessive disorder with a mutation in the 3- brain barrier (BBB) and blood-CSF barrier, using the Na+ PGDH gene, which limits L-serine biosynthesis (Acuna- dependent and independent alanine-serine-cysteine trans- Hidalgo et al. 2014). If present as a homozygous mutation, it porters (asc-1, asc-2; Kasai et al. 2011; Fuchs et al. 2011). causes prenatal or early postnatal death due to severe Biosynthesis in the glial cells likely plays a large role in pro- malformations, including microcephaly, limb malformations, viding serine for the CNS (de Koning and Klomp 2004). and sometimes neural tube defects, suggesting the importance of L-serine in fetal growth and development (Acuna-Hidalgo et al. 2014). Defects in the L-serine biosynthetic pathway dur- 3-phosphoglycerate ing infancy or childhood can lead to microcephaly, develop- mental and intellectual disabilities, and epileptic seizures, NAD+ 3-phosphoglycerate among other symptoms (El-Hattab 2016). Treatment with L- dehydrogenase (3-PGDH) serine in these diseases at concentrations of 200–700 mg/kg/ NADH+ day supplemented with glycine in some trials (200–300 mg/ H+ kg/day) resulted in some improvement of symptoms (de- 3-phosphohydroxypyruvate creased seizures, reduced spasticity, increased white matter volume, and myelination), along with boosts in behavior and 3-phosphoserine school performance (Tabatabaie et al. 2011;El-Hattab2016). GLU aminotransferase (PSAT) Unfortunately, there was no improvement of psychomotor de- KET velopment with L-serine supplementation (de Koning et al. 3-phosphoserine 2002; de Koning et al. 2004), suggesting serine deficiency during development mediates lasting neurological damage.

3-phosphoserine phosphatase (PSPH) H2O In Vitro Studies Pi L-Serine Is a Critical Component of Cell Cultures and Is Neurotrophic

L-serine L-serine is known to have important functions in the mamma- Fig. 1 Enzymes involved in the synthesis of L-serine. GLU glutamate, lian CNS other than being a constituent of proteins. It is the KET α-ketoglutarate, Pi inorganic phosphate7 metabolic precursor of neuroactive substances, D-serine, and 216 Neurotox Res (2018) 33:213–221 glycine (Verleysdonk and Hamprecht 2000), and serves as a Taken together, these observations support the need for L- building block for phosphatidylserine and sphingolipids, both serine supplementation in cell culture media, in particular for important components of the plasma membrane. Glycine is neuronal cultures, and provide evidence that L-serine func- synthesized from L-serine via a one-step reaction in the pres- tions as a conditional non-essential amino acid. ence of the enzyme, serine hydroxymethyltransferase, and is required for the synthesis of glutathione and purine (Newman L-Serine as an Important Site and Magasanik 1963). Therefore, L-serine and glycine may for Phosphorylation—Relevance of L-Serine play essential roles in supporting neuronal survival other than as a Therapeutic acting as a substrate for protein synthesis (Yang et al. 2000). L-serine has a marked effect on the survival of the pheno- Apart from playing a role in development, neuritogenesis, and type of a variety of cells in culture (Savoca et al. 1995)which survival, L-serine also functions as a component of proteins in supports its classification as a conditional non-essential amino mature cells. Amino acid residues are important sites for phos- acid. As early as 1959, it was reported that HeLa cells had phorylation with over 95% occurring at L-serine residues, 3– greater plating efficiency in minimal growth medium when 4% on L-threonine residues, and less than 1% on L-tyrosine supplemented with L-serine alone and in the absence of all residues (Nestler and Greengard 1999). Post-translational other amino acids (Lockart and Eagle 1959). Early studies modification by phosphorylation is one of the most prevalent looking at the growth of chicken embryonic dorsal root gan- modifications that occur in a cell, and at any one time, as much glion (DRG) neurons in vitro, reported that L-serine at as one third of the total human proteome may be undergoing 100 μM increased neuronal length and branching when com- phosphorylation or dephosphorylation (Cohen 2002). The ad- pared to culture media where no L-serine was present (Savoca dition or removal of phosphate residues is fundamental to et al. 1995). This effect was concentration-dependent, stereo- mediating diverse functions including cell cycle, apoptosis, specific, and mediated by L-serine rather than a derivative or and growth and signal transduction (Hunter 2012). metabolic product, as determined by a lack of effect when L- Phosphorylation acts as a fast, reversible, and highly specific serine peptides were added instead. process in order to temporarily induce or inhibit protein func- Further evidence for L-serine’s stereospecific effect was tion. Changes in phosphorylation status have the ability to observed in cultured Purkinje neurons (PN), where the sup- alter protein interactions, change protein conformation, and plementation of growth medium with both L-serine (100 μM) label proteins for degradation and removal (Perluigi et al. and glycine (40 μM) showed a greater improvement in PN 2016) (for a review see Humphrey et al. 2015). A network survival compared to medium deficient in both amino acids. of 500 kinases and 100 phosphatases have so far been char- D-serine, however, did not support PN survival, indicating acterized as regulators of protein phosphorylation (Huttlin that the pro-survival effect of serine is stereospecific (Furuya et al. 2010), and dysfunctional phosphorylation, particularly et al. 2000). of tau [for a review see (Iqbal et al. 2016)] is regarded as one L-serine also significantly promotes the maturation of mem- of the crucial steps leading to neurodegenerative disease brane voltage responses. Where PN grown in L-serine supple- onset. mented media discharge actively in response to depolarizing As already stated, L-serine is susceptible to substitution by currents, neurons grown in L-serine-deficient media exhibit on- non-protein amino acids during normal protein synthesis ly transient firing (Furuya and Watanabe 2003). (Dunlop et al. 2013;Gloveretal.2014) if they are present in In a study of cultured rat cerebrocortical neurons, L-serine the amino acid pool. Indeed, toxicity associated with the promoted survival in a concentration-dependent manner, con- misincorporation of non-protein amino acids, in both humans comitant with the upregulation of the pro-survival gene prod- and animals, has been known for decades (Dunlop et al. uct, BCL2L2 (or Bcl-w) (Yang et al. 2000). Expression of this 2014). Given that L-serine residues serve as a primary site gene has been shown to contribute to reduced cell apoptosis for phosphorylation – andthatthisreversiblereactioniscrit- under cytotoxic conditions (Gibson et al. 1996). The authors ical for cell function and homeostasis – it is crucial to maintain plated neurons from the cerebral cortices of 17-day-old rat sufficient concentrations of L-serine within the cell. This embryos and reported that when cultured in the absence of could be aided by L-serine supplementation, which could play L-serine and glycine, the cells gradually degenerated. a role in neuroprotection. However, with the addition of L-serine or L-serine in combi- Dysregulation of phosphorylation is associated with pro- nation with glycine, survival was significantly enhanced, as gressive neurodegenerative diseases in humans, including measured by an Alamar blue assay (Yang et al. 2000). An Alzheimer’s disease (AD) (Perluigi et al. 2016), explanation for the latter observation came when it was dis- frontotemporal lobar degeneration (FTLD), frontotemporal covered that neurons have a requirement for exogenously sup- lobar degeneration with motor neuron disease (FTLD- plied L-serine, delivered from astrocytes and glia (Furuya MND), frontotemporal lobar degeneration with ubiquitinated et al. 2000). inclusions (FTLD-U) (Hasegawa et al. 2008), and ALS Neurotox Res (2018) 33:213–221 217

(Hasegawa et al. 2008). Hundreds of L-serine phosphoryla- 2004). p62 has an important role in protein degradation since tion reactions are already classified in the literature but we will it contains a ubiquitin-associated domain at the C-terminus limit our discussion to L-serine phosphorylation or dysregu- that interacts with ubiquitinated and misfolded proteins. By lation as it is relevant to neurodegeneration and also interacting with autophagy-related gene 8 (ATG8), it di- neuroprotection. rects misfolded proteins to autophagosomes for eventual lyso- somal degradation. In addition, interaction with the protea- L-Serine Phosphorylation in Progressive some subunit regulatory particle 1 (Rpt1) favors degradation Neurodegenerative Disease by the proteasome. p62-mediated selective autophagy is en- hanced by phosphorylation of serine403 (pS403) and has been TAR DNA binding protein of 43 kDa (TDP-43) is a major implicated in progressive neurodegenerative disease, since protein associated with ALS (Neumann et al. 2006). It can be pS403 p62 accumulates in AD and ALS-diseased tissue found associated with ubiquitin immunoreactive neuronal cy- (Kurosawa et al. 2016). Also, phosphorylation of serine349 toplasmic inclusions in several disorders including FTLD-U, (pS349) on p62 has been implicated in AD since the ratio of FTLD-MND, and ALS, which are collectively referred to as pS349 to total p62 levels increases significantly in AD brains TDP43-proteinopathies (Hasegawa et al. 2008). compared to controls (Tanji et al. 2014). In ALS and FTLD-U, abnormal C-terminal fragments of Phosphorylation and dephosphorylation of L-serine in pro- TDP-43 are ubiquitinated, hyperphosphorylated, and accumu- teins can also constitute a protective mechanism which regu- late as cytoplasmic inclusions in neurons and glia. In a non- lates recovery of cells from endoplasmic reticulum (ER) pathological state, TDP-43 functions in regulating transcrip- stress, the latter of which is linked to neurodegeneration. tion and alternative splicing and is located in the nucleus. The unfolded protein response (UPR) is an evolutionarily However, cells with cytoplasmic inclusions of TDP-43 have conserved response to ER stress, comprised of three trans- been demonstrated to show an absence of the normal nuclear membrane sentinels : PKR-like ER kinase (PERK), inositol- TDP-43 localization (Mackenzie and Rademakers 2008). requiring enzyme 1 (IRE1), and activating transcription factor There are multiple potential phosphorylation sites within 6 (ATF6). IRE1 is activated by auto phosphorylation on ser- human TDP-43, including 41 serine (S) residues. Hasegawa ine724, leaving the endonuclease activity free to cleave X-box et al. (2008) generated 39 different synthetic TDP-43 binding protein-1 (XBP1) to the spliced and active transcrip- phosphopeptides, then raised antibodies representing 36 out tion factor, spliced XBP1 (XBP1s). XBP1s is a highly active of 63 candidate phosphorylation sites, and reported that phos- transcription factor and acts in concert with ATF6 to activate phorylated S379 (pS379), pS403/404, pS409, pS410, and the transcription of genes encoding for ER chaperone proteins, pS409/410 show intensely immunostained ubiquitin-positive folding enzymes, and components of the endoplasmic- inclusions (UPIs) in FTLD-U and ALS. reticulum-associated protein degradation (ERAD) machinery Immunohistochemical staining showed that five of the (Yamamoto et al. 2007), which functions to reinstate homeo- phosphorylation-specific anti-TDP-43 antibodies identified stasis during ER stress (Lee et al. 2003). UPIs in both FTLD-U and ALS brains. The activation of PERK results in the addition of a phos- In the brain, the transition from presymptomatic AD to a phate group to serine51 on the alpha subunit of eukaryotic symptomatic state correlates with increased density of NFTs initiation factor 2 (eIF2α) and serves as a signal to inhibit and accumulating amyloid β-peptide (Nelson et al. global protein synthesis. This is considered to be a rate- 2012; Perluigi et al. 2016). Changes in cognitive decline and limiting step in mRNA translation and a switch for cells to short-term memory in AD are, therefore, strongly correlated clear accumulated misfolded proteins and thus, overcome with tau hyperphosphorylation (Nelson et al. 2012). Thus, it is proteotoxic stress (Wek et al. 2006 ). In eukaryotic cells, eu- suggested that these changes promote a transition to AD. For karyotic initiation factor 2 (eIF2), together with the initiator this reason, extensive analysis of the AD phosphoproteome methionyl tRNA (Met-tRNAi) and guanosine triphosphate has been undertaken, confirming L-serine (GTP), forms a ternary complex (eIF2.GTP.Met-tRNAi) and hyperphosphorylation is associated with neuropathology. phosphorylation of eIF2α by any one of the four kinases— Furthermore, studies in yeast transfected to express mutants general control nonderepressible 2 (GCN2), PERK, protein of human protein tau typical of frontotemporal dementia with kinase R (PKR), and hepatic heme-regulated inhibitor parkinsonism (FTDP), showed that pS409 is a direct determi- (HRI)—negatively affects the guanosine diphosphate- nant for tau aggregation (Vanhelmont et al. 2010). (GDP)-GTP exchange activity of the β-subunit of eIF2 p62/SQSTM1/sequestosome 1 (known as p62) is a multi- (Sonenberg and Hinnebusch 2009). This prevents the functional degradation protein that has been found phosphor- recycling of eIF2 between successive rounds of protein syn- ylated and co-localized with ubiquitin-tau-containing inclu- thesis (Yang and Hinnebusch 1996), thus reducing global pro- sions in the hippocampus and neocortex of patients with tein translation and favoring degradation of damaged/ AD, but not age-matched controls (Seibenhener and Babu misfolded proteins. This process is relevant for 218 Neurotox Res (2018) 33:213–221 neurodegenerative disease as it may favor recovery of neurons to those subjects fed L-BMAA alone (Cox et al. 2016). These from ER stress as a result of the deposition of misfolded/ data support the hypothesis that L-serine is neuroprotective. ubiquitinated/hyperphosphorylated proteins. Sustained activa- tion of this pathway, however, eventually targets cells for ap- optosis, and for this reason, ER stress is implicated in cell Clinical Trials and Therapeutic Potential death in several types of neurodegenerative disease. The interplay between protein-bound L-serine, phosphory- Since L-serine is synthesized in situ, effects on its metabolism lated serine, and how it relates to neurodegeneration, is com- may offer insights into human disease and potentially thera- plex and apparently paradoxical. As we have already present- peutic targets. Dysregulation of L-serine synthesis and ed, in some cases, post-translational modification of serine metabolic pathways have been associated with a number of residues is protective, as in the phosphorylation of serine51 human conditions and have been reviewed extensively by α on eIF2 , and in other cases, its presence in plaques and NFTs Nishikawa (2011) and Canu et al. (2014). suggests a role in pathology. However, these observations should not be confused with L-serine as a free amino acid supplement, which shows virtually no toxicity, and indeed Metabolic has been shown to be neuroprotective, as discussed below. Excessive alcohol consumption can result in fatty liver dis- ease, which untreated can ultimately lead to liver failure and In Vivo Studies death (Toshikuni et al. 2014). In mice experimentally dosed with ethanol, both homocysteine and hepatic triglycerides Experimental research examining the impacts of dispropor- were increased 5-fold. These effects were ameliorated by die- tionate concentrations of individual amino acids began in ear- tary L-serine supplementation (200 mg/kg) with decreasing nest in the 1940s. Initial research on the conditional non- hepatic neutral lipid accumulation and increased glutathione essential amino acid L-serine, primarily investigated racemic and S-adenosylmethionine (Sim et al. 2015). mixtures of toxicity to DL-serine and L-serine, identifying pure forms of L-serine to be non-toxic (Artom et al. 1945; Neurological Hardy et al. 1960). Since D-serine is biologically very differ- ent from L-serine, in both form and function, and our interest For human consumption, the US FDA has classified L-serine here is purely in the L-form, we here discuss only L-serine. as GRAS for use as a food additive, as long as it does not From these early studies, several facts became apparent exceed 8.4% of total protein in the diet (CFR Title 21 which add context to our current understanding of L-serine Section 17.320.18). supplementation as a therapeutic approach. Rats given a single L-serine has previously been used to treat polyneuropathy dose of L-serine (approximately 1 to 1.3 g/kg) did not show (Catsman-Berrevoets et al. 1997) and is currently being eval- adverse effects (Artom et al. 1945; Hardy et al. 1960). uated for use in other neurological conditions. Low L-serine Excessive doses of L-serine, 20–60 times that of these early concentrations were documented in a girl displaying progres- studies, indicated that at these concentrations, representing sive polyneuropathy alongside a number of other conditions 2.1, 4.2, and 6.3% of the total protein intake (21–63 g/kg), such as ichthyosis. Treatment with L-serine at up to 400 mg/ L-serine decreased weight in rats (by 6, 25, and 29%, respec- kg/day for 3 weeks resulted in amelioration of these symptoms tively) compared to control animals fed a standard basal diet (Catsman-Berrevoets et al. 1997). (Benevenga and Harper 1967). These same concentrations, Patients with essential tremors exhibit decreased concen- however, alleviated some of the toxic effects of both high trations of L-serine in both blood serum and CSF, which sug- methionine (3%) and high homocysteine (3%) in the diet gests abnormalities in L-serine biosynthetic pathways (Mally (Benevenga and Harper 1967). In summary, L-serine is non- et al. 1996). Hereditary sensory neuropathy type 1 (HSN-1) is toxic to rats, but at excessive concentrations exceeding 21 g/ a dominantly inherited disease that leads to progressive de- kg, induced a loss in weight. generation of DRG and motor neurons (Hirabayashi and Non-human primates, Chlorocebus sabaeus, were fed a Furuya 2008). The disease has been directly linked with de- low protein diet supplemented with 210 mg/kg/d L-serine, fects in sphingolipid biosynthesis, and current therapeutic with and without the neurotoxin L-BMAA (effective daily studies are underway to evaluate the effect of L-serine supple- dose of L-BMAA was 160 mg/kg), for a total of 140 days. mentation on disease outcomes. Harvard University and Animals fed L-serine alone showed no adverse effects and Massachusetts General Hospital conducted a 2-year study on were indistinguishable from control animals (Cox et al. L-serine supplementation to correct biochemistry associated 2016). Animals co-administered L-serine with L-BMAA re- with HSN-1. In this study of 14 subjects, L-serine, given at sulted in a 35–85% reduction in the density of NFTs compared either 200 or 400 mg/kg (roughly 15 and 30 g/day, Neurotox Res (2018) 33:213–221 219 respectively) showed no adverse effects with either dose after exclusion criteria as our phase I trial in an attempt to further 10 weeks of therapy (Garofalo et al. 2011). demonstrate the tolerability of L-serine at 30 g/day and to In a comprehensive ethnobotanical survey of the diet of replicate preliminary indications of efficacy that were detected centenarians and other aged residents of Ogimi village in in the small phase I trial (Levine et al. 2017). Okinawa, known throughout Japan as Blongevity village,^ we found that traditional food items rich in L-serine, including tofu (made from fermented soy beans), edamame, a variety of Conclusion seaweeds, sweet potatoes, and pork, constitute the most fre- quently consumed items. On average, Ogimi villagers are re- L-serine which has traditionally been considered to be a non- ceiving 3–4 times the amount of L-serine as their American essential amino acid may play a more important role in neu- counterparts. Since there is an apparent paucity of progressive rological development than historically realized, with reliance neurodegenerative illness among the Ogimi villagers, it has on endogenous L-serine in the diet becoming of increased been hypothesized that L-serine in the traditional Ogimi diet interest. L-serine has a variety of roles and functions from serves as a neuroprotective substance (Cox and Metcalf 2017). primary protein structure to cell signaling, the latter primarily A phase I human clinical trial which we initiated for the via post-translational modification by phosphorylation. safety of L-serine for ALS patients (NCT01835782) was re- Deficiencies in L-serine may have profound health effects cently published (Levine et al. 2017). Patients (n = 20) were from embryo to geriatrics. Experimental investigations with given either 1, 5, 15, or 30 g/day of L-serine for 6 months with animals and humans support the FDA determination that L- very few side effects reported [bloating, nausea, and loss of serine is GRAS. Dietary supplementation with L-serine is now appetite; (Levine et al. 2017)]. Analysis of the ALSFRS-R being clinically investigated as a possible therapy for progres- slopes indicated that ALS patients taking L-serine had a re- sive neurodegenerative diseases including ALS and duction in the rate of functional loss as compared to historical Alzheimer’sdisease. ALS control patients (five studies that included patients with symptom duration < 3 years and functional vital capacity Compliance with Ethical Standards (FVC) ≥ 60% to compare with the L-serine patients). There was no evidence of neurological harm to ALS patients as Competing Interests The Institute for Ethnomedicine has applied for patents for the use of L-serine to treat neurodegenerative illness (US 13/ compared to controls, and this study concluded that L-serine 683,821). at doses up to 30 g/day is safe for ALS patients (Levine et al. 2017). In 2017 there are three human clinical trials evaluating the References use of L-serine as a therapeutic agent (Table 1). Two of these trials, listed on clinicaltrials.gov, involve neurological Acuna-Hidalgo R, Schanze D, Kariminejad A et al (2014) Neu-Laxova conditions and one involves fatty liver disease: (1) syndrome is a heterogeneous metabolic disorder caused by defects NCT01733407, 400 mg/kg/d L-serine, BL-Serine in enzymes of the L-serine biosynthesis pathway. 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